Cellular characterization of pituitary adenoma cell line (AtT20 cell) transfected with insulin, glucose transporter type 2 (GLUT2) and glucokinase genes: insulin secretion in response to physiological concentrations of glucose

We investigated the mechanisms of insulin secretion by transfecting into a pituitary adenoma cell line (AtT20) a combination of genes encoding human insulin (HI), glucose transporter type 2 (GLUT2) and glucokinase (GK), followed by studying the characteristics of these cells. In static incubation, a cell line transfected with insulin gene alone (AtT20HI) secreted mature human insulin but this was not in a glucose-dependent manner. Other cell lines transfected with insulin and GLUT2 genes (AtT20HI-GLUT2-3) or with insulin and GK genes (AtT20HI-GK-1) secreted insulin in response to glucose concentrations of only less than 1 mmol/l. In contrast, cell lines transfected with insulin, GLUT2 and GK genes (AtT20HI-GLUT2-GK-6, AtT20HI-GLUT2-GK-7 and AtT20HI-GLUT2-GK-10) showed a glucose-dependent insulin secretion up to 25 mmol/l glucose. Glucose utilization and oxidation were increased in AtT20HI-GLUT2-GK cell lines but not in AtT20HI, AtT20HI-GLUT2-3 and AtT20HI-GK-1 cells at physiological glucose concentrations, compared with AtT20 cells. Diazoxide, nifedipine and 2-deoxy glucose suppressed (p < 0.05) glucose stimulated insulin secretion in AtT20HI-GLUT2-GK-6 cells. Glibenclamide, KCl or corticotropin releasing factor (CRF) stimulated (p < 0.05) insulin secretion both in AtT20HI and AtT20HI-GLUT2-GK-6 cells. Insulin secretion stimulated by glibenclamide, KCl or CRF was further enhanced by the addition of 25 mmol/l glucose in AtT20HI-GLUT2-GK-6 cells but not in AtT20HI cells. In perifusion experiments, a stepwise increase in glucose concentration from 5 to 25 mmol/l stimulated insulin secretion in AtT20HI-GLUT2-GK cell lines but the response lacked a clear first phase of insulin secretion. Our results suggest that both GLUT2 and glucokinase are necessary for the glucose stimulated insulin secretion in at least rodent cell lines, and that other element(s) are necessary for a biphasic insulin secretion typically observed in beta cells.     

Temporal and quantitative correlations between insulin secretion and stably elevated or oscillatory cytoplasmic Ca2+ in mouse pancreatic beta-cells

An increase in cytoplasmic Ca2+ in beta-cells is a key step in glucose-induced insulin secretion. However, whether changes in cytoplasmic free Ca2+ ([Ca2+]i) directly regulate secretion remains disputed. This question was addressed by investigating the temporal and quantitative relationships between [Ca2+]i and insulin secretion. Both events were measured simultaneously in single mouse islets loaded with fura-PE3 and perifused with a medium containing diazoxide (to prevent any effect of glucose on the membrane potential) and either 4.8 or 30 mmol/l K+. Continuous depolarization with 30 mmol/l K+ in the presence of 15 mmol/l glucose induced a sustained rise in [Ca2+]i and insulin release. No oscillations of secretion were detected even after mathematical analysis of the data (pulse, spectral and sample distribution analysis). In contrast, alternating between 30 and 4.8 mmol/l K+ (1 min/2 min or 2.5 min/5 min) triggered synchronous [Ca2+]i and insulin oscillations of regular amplitude in each islet. A good correlation was found between [Ca2+]i and insulin secretion, and it was independent of the presence or absence of oscillations. This quantitative correlation between [Ca2+]i and insulin secretion was confirmed by experiments in which extracellular Ca2+ was increased or decreased (0.1-2.5 mmol/l) stepwise in the presence of 30 mmol/l K+. This resulted in parallel stepwise increases or decreases in [Ca2+]i and insulin secretion. However, while the successive [Ca2+]i levels were unaffected by glucose, each plateau of secretion was much higher in 20 than in 3 mmol/l glucose. In conclusion, in our preparation of normal mouse islets, insulin secretion oscillates only when [Ca2+]i oscillates in beta-cells. This close temporal relationship between insulin secretion and [Ca2+]i changes attests of the regulatory role of Ca2+. There also exists a quantitative relationship that is markedly influenced by the concentration of glucose.     

The biguanide compound metformin prevents desensitization of human pancreatic islets induced by high glucose

Pancreatic islet desensitization by high glucose concentrations is a temporary and reversible state of beta-cell refractoriness to glucose (and possibly other secretagogues), due to repeated or prolonged pre-exposure to increased glucose concentrations. We evaluated whether the oral antidiabetic agent metformin affects this phenomenon in isolated, human pancreatic islets, and whether the possible effects of the biguanide are influenced by the presence of a sulphonylurea, glyburide. Islets prepared from five human pancreases were incubated for 24 h in M199 culture medium containing either 5.5 or 22.2 mmol/l glucose, with or without a therapeutic concentration (2.4 microg/ml) of metformin. Then, the islets were challenged with either 3.3 mmol/l glucose, 16.7 mmol/l glucose, or 3.3 mmol/l glucose + 10 mmol/l arginine, and insulin release was measured. After incubation in the absence of metformin, the human islets exposed to 22.2 mmol/l glucose showed no significant increase in insulin release when challenged with 16.7 mmol/l glucose (confirming that hyperglycemia desensitizes pancreatic beta-cells). In the presence of metformin, the islets fully maintained the ability to significantly increase their insulin release in response to glucose, even when previously exposed to 22.2 mmol/l glucose. No major effect on arginine-induced insulin release was observed, whatever the culture conditions. The protective action of metformin was observed also when glyburide was present in the incubation medium, whereas the sulphonylurea alone did not affect insulin release from the islets previously exposed to high glucose concentrations. These in vitro results suggest that metformin can prevent the desensitization of human pancreatic islets induced by prolonged exposure to increased glucose concentrations.     

Medical, morphological and functional aspects of Greek football referees

Though the pig appears to be the islet donor of choice for grafts in diabetic patients, there may be a risk of transmission of infectious agents. In this context, we adopted a strategy of islet isolation from pigs raised and killed in specific pathogen-free (SPF) conditions as a minimum with regard to the concept of quality assurance. Accordingly, the present study investigated the function of SPF pig islets to determine whether they react qualitatively and quantitatively to nutriments, hormones and neuromediators with which they would be confronted in man and could therefore provide effective regulation during physiologic or physiopathologic situations. beta cells from 18 Large-White SPF pigs were functionally intact after 7 days in culture. Insulin stimulation indexes (SI) of 3.1 +/- 0.2, 2.2 +/- 0.1, and 4.4 +/- 0.3 were found respectively for 30 mmol/l K+, 100 mumol/l tolbutamide and 10 mmol/l theophylline. Basal insulin secretion (72.2 +/- 7.6 muU/min) had already increased significantly (p < 0.001) with 5.5 mmol/l glucose (184.2 +/- 25.5 muU/min, SI: 2.5 +/- 0.6), indicating that the threshold stimulatory concentration was comparable to that of human islets. Insulin secretion increased in a glucose dose-dependent manner (p < 0.001): SI: 3.1 +/- 0.3 and 3.6 +/- 0.2 with 11.0 mmol/l and 22.0 mmol/l glucose, which showed a satisfactory magnitude with reference to human islets. Even the subtle phenomenon of "glucose memory" was apparent in these pig islets. Arginine stimulated (p < 0.001) insulin secretion dose-dependently (SI: 2.2 +/- 0.3 with 5 mmol/l and 2.9 +/- 0.2 with 10 mmol/l). The ketone body beta-hydroxybutyrate (10 mmol/l) also induced insulin secretion (SI: 4.3 +/- 0.3). Insulin release was stimulated by 4 mumol/l gastric inhibitory peptide, revealing sensitivity to the hormonal enteroinsular axis, and by 2 mumol/l glucagon. Parasympathetic cholinergic influence was studied using 500 mumol/l carbamylcholine, which increased insulin secretion. The influence of orthosympathetic control and of stress situations was also studied. As in human islet response, epinephrine and the alpha 2-agonist clonidine (50 mumol/l) inhibited insulin secretion. Finally pre-culture of islets may be beneficial for graft outcome, provided that no deterioration in islet function occurs. A prolonged 21-day culture of SPF pig islets showed no decrease in insulin response to glucose, arginine and potassium, even with an unaltered threshold stimulatory glucose concentration. Thus, Large-White SPF pigs and the application of our isolation procedure provided islets with functional characteristics reproducibly compatible with potential utilisation for effective regulation of glycaemia under physiologic and physiopathologic situations in humans.     

Insulin secretion in pancreatic islets from rats with cirrhosis

Cirrhosis was induced in rats by subcutaneous injections of CCl4 for 13 or 17 weeks. The morphology of the pancreatic islets from the CCl4-treated rats was found to be normal. The CCl4-treated rats had lower fasting serum glucose levels and higher serum insulin levels than the controls. After an oral glucose load (3 g/kg body weight), glucose levels in CCl4-treated rats stayed within the normal range, whereas the serum insulin levels remained higher with a delayed decline of insulin with time. In vitro perifusion of islets from the CCl4-treated rats showed that the response to 16.7 mmol/l glucose was reduced with both lower total insulin output and stimulated insulin output, whereas the patterns of first and second phase of insulin release did not differ. The insulin content of the perifused islets was not affected by 13 weeks of CCl4 treatment. Islets from rats treated with CCl4 for 17 weeks showed normal secretory response to 20 mmol/l L-arginine. Taken together, the results, showing normal or reduced capacity for insulin secretion, suggest that the hyperinsulinemia accompanying CCl4-induced cirrhosis is not due to increased secretion of the pancreatic islets. It may rather be associated with decreased insulin degradation by the liver with cirrhosis.     

The priming effect of glucose on insulin secretion from isolated islets of Langerhans

Biphasic insulin secretion from perifused rat islets of Langerhans was enhanced if islets had previously been stimulated with glucose 16.6 mmol/l. The priming effect of glucose was reduced if mannoheptulose (16.6 mmol/l), deuterium oxide (D2O; 98% v/v) or adrenaline 10 mumol/l) was included in the medium during the initial stimulation period, or if Calcium was omitted. Glyceraldehyde (16.6 mmol/l) but not theophylline (5 mmol/l) could substitute for glucose during the initial stimulation and make islets more responsive to subsequent stimulation. The results suggest that the priming effect of glucose on insulin secretion may be related to 1) glucose metabolism and 2) Ca fluxes in the B cell and the consequent activation of the microtubular system. Neither the generation of intracellular cyclic AMP nor the release of insulin per se appears to be involved in the priming process.     

Interaction of glucose and metformin with isolated red cell membrane

Isolated human erythrocyte membranes (red blood cell (RBC) ghosts) were incubated with glucose at 5, 10, 20 and 100 mmol/l concentrations, with insulin (0.01 to 200 mU/l) and metformin (CAS 657-24-9) 0.5 up to 50.0 mumol/l). Binding studies with 14C-glucose and subsequent gel electrophoresis revealed 60% of the radioactivity around ban 4.2-4.5 at 5 mmol/l, whereas a random distribution of radioactivity over all protein bands of the RBC membrane was found at 20 mmol/l concentration after incubation for 30 min or 48 h. Metformin does not bind covalently to RBC membranes, however, after photochemical linkage of 14C-metformin via the aminoreactive linker azidophenylglyoxal the highest radioactivity (21%) was counted in the range of band 4.2-4.5. In parallel with an increase of order parameters of 5-doxyl-stearic acid the thiol status of the membranes decreases as determined by monobromobimane fluorescence. 20 and 100 mmol/l concentrations of glucose decrease the reactivity of membrane thiols towards bromobimane significantly to 73 and 62% of the controls. Concomitantly, membrane fluidity at polar sites is diminished as measured by order parameters of spin label 5-doxyl stearic acid. In RBC membranes pretreated with 20 mmol/l glucose the decreased fluorescence is significantly raised again by insulin and metformin. This effect is even more pronounced, if insulin and metformin are incubated together. Reaction of membrane thiols with a maleimido spin label detects modification in the ratio of mobile and immobilized spin label populations in the electron paramagnetic resonance signal under the above conditions, indicative of conformational changes of membrane proteins.     

Role of cAMP in upregulation of insulin secretion during the adaptation of islets of Langerhans to pregnancy

Islets undergo a number of upregulatory changes to meet the increased demand for insulin during pregnancy, including an increase in glucose-stimulated insulin secretion with a reduction in the stimulation threshold. Treatment with the lactogenic hormone prolactin (PRL) in vitro has been shown to induce changes in islets similar to those observed during pregnancy. We examined cAMP production in islets treated with PRL to determine if changes in cAMP are involved in the upregulation of insulin secretion. Insulin secretion and cAMP concentrations were measured from islets in response to a suprathreshold (6.8 mmol/l) or high (16.8 mmol/l) glucose concentration in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Insulin secretion increased by 2.1-, 5.0-, and 5.9-fold at the suprathreshold glucose concentration and by 1.6-, 2.3-, and 2.9-fold at the higher glucose concentration after 1, 3, and 5 days of PRL treatment, respectively. After a similar pattern, cAMP metabolism increased by 1.2-, 1.6-, and 2.1-fold at the suprathreshold glucose concentration and by 1.2-, 1.7-, and 2.2-fold at the high glucose concentration after 1, 3, and 5 days of PRL treatment, respectively. The similar increases in insulin secretion and cAMP concentration suggest that changes in cAMP metabolism are involved in lactogen-induced upregulation of insulin secretion. To gain additional insight into the role of cAMP in the upregulation of islet function after lactogen treatment, we examined the relationship between changes in cAMP concentration and insulin secretion. Under all conditions (differing glucose concentrations and time periods), the increase in insulin release was directly proportional to the increase in cAMP. Thus increased glucose-stimulated insulin secretion from lactogen-treated islets could be accounted for by increased generation of cAMP and did not appear to require any further specific changes in intracellular processes mediated by cAMP. Because the PRL receptor is not directly involved in cAMP metabolism, the lactogen-induced increase in cAMP was most likely due to the increase in glucose metabolism that we have previously demonstrated in PRL-treated islets and in islets during pregnancy.     

Enhanced glucose-dependent insulinotropic polypeptide secretion and insulinotropic action in glucagon-like peptide 1 receptor -/- mice

Incretins are gastrointestinal hormones that act on the pancreas to potentiate glucose-stimulated insulin secretion. Despite the physiological importance of the enteroinsular axis, disruption of glucagon-like peptide (GLP)-1 action is associated with only modest glucose intolerance in GLP-1 receptor -/- (GLP-1R -/-) mice. We show here that GLP-1R -/- mice exhibit compensatory changes in the enteroinsular axis via increased glucose-dependent insulinotropic polypeptide (GIP) secretion and enhanced GIP action. Serum GIP levels in GLP-1R -/- mice were significantly elevated versus those in +/+ control mice after an oral glucose tolerance test (369 +/- 40 vs. 236 +/- 28 pmol/l; P < or = 0.02). Furthermore, GIP perfusion of mice pancreas and isolated islets in the presence of elevated glucose concentrations elicited a significantly greater insulin response in GLP-1R -/- than in +/+ mice (P < or = 0.02-0.05). In contrast, no significant perturbation in the insulin response to perfused glucagon was detected under conditions of low (4.4 mmol/l) or high (16.6 mmol/l) glucose in GLP-1R -/- mice. Total pancreatic insulin but not glucagon content was significantly reduced in GLP-1R -/- compared with in +/+ mice (77 +/- 9 vs. 121 +/- 10 pmol/mg protein; P < or = 0.005). These observations suggest that upregulation of the GIP component of the enteroinsular axis, at the levels of GIP secretion and action, modifies the phenotype resulting from interruption of the insulinotropic activity of GLP-1 in vivo.     

Effect of a non-sulphonylurea hypoglycaemic agent, KAD-1229 on hormone secretion in the isolated perfused pancreas of the rat

1. We examined the cooperative effect of a newly synthesized oral hypoglycaemic agent, KAD-1229 with glucose on insulin, glucagon and somatostatin secretion in the isolated perfused pancreas of the rat. 2. KAD-1229 stimulated concentration-dependently the first phase of insulin secretion without the second phase in the presence of 2.8 mM glucose, while it stimulated both the first and the second phase of insulin release in the presence of 5.6 mM glucose. It was confirmed that the first phase of insulin release is depolarization-induced release with no other additional signal transduction. 3. KAD-1229 also enhanced insulin release evoked by 16.7 mM glucose, a concentration known to inhibit the ATP-sensitive K+ current completely. 4. A low concentration (2.8 mM) of glucose stimulated somatostatin release transiently, while a higher concentration (16.7 mM) of glucose exerted a sustained stimulation. KAD-1229 stimulated somatostatin secretion in a concentration-dependent manner irrespective of glucose concentrations. 5. When glucagon release was stimulated with 2.8 mM glucose, KAD-1229 inhibited this hypoglycaemia-induced glucagon secretion. 6. When pancreata from rats pretreated with streptozotocin (STZ) 60 mg kg-1 were perfused, the basal secretion of glucagon was markedly elevated, and the glucagon response to the low glucose was abolished. Further, the insulin and somatostatin responses to KAD-1229 were largely attenuated. KAD-1229 showed transient enhancement followed by inhibition of the glucagon release from the STZ-pretreated rat pancreas. 7. We conclude that KAD-1229 stimulates insulin and somatostatin release, while it inhibits glucagon release following transient stimulation.     

Glucose-dependent arginine stimulation test for characterization of islet function: studies on reproducibility and priming effect of arginine

Quantitative determination of insulin secretion is of importance both clinically and in research. The optimal method has not been established, although several different methods have been used. We determined the reproducibility of islet function parameters obtained by the glucose-dependent arginine stimulation test, and also studied the priming effect of arginine on subsequent acute insulin responses. The test measures the acute insulin (AIR) and glucagon (AGR) responses to i.v. arginine (5 g injected over 45 s) at fasting glucose and glucose concentrations clamped at 14 and above 25 mmol/l, as well as the glucose potentiation of insulin secretion (slopeAIR) and the glucose inhibition of glucagon secretion (slopeAGR). When the test was performed twice in seven healthy women (mean +/- SD age 58.7 +/- 0.5 years, BMI 27.6 +/- 5.5 kg/m2), the AIRs to arginine had a within-subject coefficient of variation (CV) of 18.6% at fasting glucose, 18.7% at 14 mmol/l glucose and 16.3% at above 25 mmol/l glucose. The CVs for AGR were 11.6, 14.9 and 8.9%, respectively. The CV of the slopeAIR was 24% and of the slopeAGR 17.2%. The arginine priming study was performed in six healthy women (age 63.7 +/- 0.3 years, BMI 28.0 +/- 6.9 kg/m2). Saline or arginine (5 g) was injected at fasting glucose, followed by arginine (5 g) at 14 mmol/l glucose. There was no difference between the acute insulin or glucagon responses to arginine at 14 mmol/l glucose in the two conditions, suggesting that there is no priming effect of arginine on the subsequent acute insulin or glucagon responses. Therefore, this method is a good tool to determine insulin secretion as, apart from its good reproducibility, it also provides several important parameters of islet function.     

Adaptation to hyperglycemia enhances insulin secretion in glucokinase mutant mice

The present study was undertaken to test the hypothesis that exposure to high glucose concentrations enhances insulin secretion in pancreatic islets from glucokinase-deficient mice. Insulin secretion and intracellular calcium ([Ca2+]i) were measured as the glucose concentration was increased from 2 to 26 mmol/l in islets from heterozygous glucokinase (GK)-deficient mice (GK+/-) and their wild-type littermates (GK+/+). Results obtained in islets incubated in 11.6 or 30 mmol/l glucose for 48-96 h were compared. GK+/- islets that had been incubated in 30 mmol/l glucose showed improved although not normal insulin secretory and [Ca2+]i responses to the standard glucose challenge as well as an enhanced ability to sense small amplitude glucose oscillations. These effects were associated with increased glucokinase activity and protein. In contrast, exposure of GK+/+ islets to 30 mmol/l glucose increased their basal insulin secretion but reduced their incremental secretory responses to glucose and their ability to detect small amplitude glucose oscillations. Thus exposure of GK+/- islets to 30 mmol/l glucose for 48-96 h enhanced their ability to sense and respond to a glucose stimulus, whereas similar exposure of GK+/+ islets induced evidence of beta-cell dysfunction. These findings provide a mechanistic framework for understanding why glucokinase diabetes results in mild hyperglycemia that tends not to increase over time. In addition, the absence of one allele of the glucokinase gene appears to protect against glucose-induced beta-cell dysfunction (glucose toxicity).     

Glucagon-like peptide 1 improves the ability of the beta-cell to sense and respond to glucose in subjects with impaired glucose tolerance

Impaired glucose tolerance (IGT) and NIDDM are both associated with an impaired ability of the beta-cell to sense and respond to small changes in plasma glucose concentrations. The aim of this study was to establish if glucagon-like peptide 1 (GLP-1), a natural enteric peptide and potent insulin secretagogue, improves this defect. Two weight-matched groups, one with eight subjects having IGT (2-h glucose, 10.1 +/- 0.3 mmol/l) and another with seven subjects with diet-treated NIDDM (2-h glucose, 14.5 +/- 0.9 mmol/l), were studied on two occasions during a 12-h oscillatory glucose infusion, a sensitive test of the ability of the beta-cell to sense and respond to glucose. Glucose was infused with a mean rate of 4 mg x kg(-1) x min(-1), amplitude 33% above and below the mean rate, and periodicity of 144 min, with infusion of saline or GLP-1 at 0.4 pmol x kg(-1) x min(-1) for 12 h. Mean glucose levels were significantly lower in both groups during the GLP-1 infusion compared with during saline infusion: 9.2 +/- 0.4 vs. 6.4 +/- 0.1 mmol/l in the IGT subjects (P < 0.0004) and 14.6 +/- 1.0 vs. 9.3 +/- 0.7 mmol/l in NIDDM subjects (P < 0.0002). Despite this significant reduction in plasma glucose concentration, insulin secretion rates (ISRs) increased significantly in IGT subjects (513.3 +/- 77.6 vs. 583.1 +/- 100.7 pmol/min; P < 0.03), with a trend toward increasing in NIDDM subjects (561.7 +/- 122.16 vs. 642.8 +/- 128 pmol/min; P = 0.1). These results were compatible with enhanced insulin secretion in the presence of GLP-1. Spectral power was used as a measure of the ability of the beta-cell to secrete insulin in response to small changes in the plasma glucose concentration during the oscillatory infusion. Spectral power for ISR increased from 2.1 +/- 0.9 during saline infusion to 7.4 +/- 1.3 during GLP-1 infusion in IGT subjects (P < 0.004), but was unchanged in NIDDM subjects (1.0 +/- 0.4 to 1.5 +/- 0.6; P = 0.3). We concluded that low dosage GLP-1 improves the ability of the beta-cell to secrete insulin in both IGT and NIDDM subjects, but that the ability to sense and respond to subtle changes in plasma glucose is improved in IGT subjects, with only a variable response in NIDDM subjects. Beta-cell dysfunction was improved by GLP-1 infusion, suggesting that early GLP-1 therapy may preserve beta-cell function in subjects with IGT or mild NIDDM.     

Accidental intake of tritiated water: a cytogenetic follow-up case on translocation stability and dose reconstruction

OBJECTIVE: To evaluate the renin-aldosterone system and insulin secretion in hyperparathyroidism and their effects on blood pressure regulation. DESIGN: Studies were carried out on patients with primary hyperparathyroidism (PHPT) prior to and following removal of the parathyroid tumor. METHODS: Sixteen normotensive and euglycemic patients with PHPT were studied. The following parameters were measured: basal and stimulated plasma renin activity (PRA) and aldosterone (ALD) secretion: parathormone (PTH) and serum electrolytes. Insulin and glucose levels were measured during an oral glucose tolerance test. RESULTS: Systolic but not diastolic blood pressure showed a decrease following surgery, from 123.3+/-13.0/80+/-8.6 to 116.7+/-13.5/77.3+/-8.8 mmHg. The decrease in the systolic pressure was not clinically significant. After surgery, both the basal and stimulated PRA and ALD values decreased, and the preoperative pathological values returned to normal: PRA basal: 1.79 --> 0.70 ng/ml/h, P=0.0049; PRA stimulated: 7.76 --> 1.90 ng/ml/h, P=0.0031; ALD basal: 111.5 --> 73.0 pg/ml, P=0.0258; ALD stimulated: 392.5 --> 236.0 pg/ml, P=0.0157. The postoperative decrease in the PRA correlated with the changes in PTH levels (r=0.5442, P < 0.05, n=16) but did not correlate with the changes in serum calcium concentrations. Both the fasting and stimulated insulin levels decreased after surgery but remained within the normal range: insulin fasting: 10.2 --> 5.0 mIU/l, P=0.0218; insulin area under the curve: 5555 --> 3296 mIU/l*min, P=0.0218. There was no correlation between the changes in insulin levels and PTH or ion levels. Sodium, potassium and blood glucose levels remained unaffected by parathyroid surgery. CONCLUSIONS: In a population of normotensive hyperparathyroid patients an increased activity of the renin-aldosterone system related to PTH was found and surgery resulted in a small and insignificant decrease in blood pressure. This change was accompanied by a significant decrease in the activity of the renin-aldosterone system indicating the role of the renin-aldosterone system in the regulation of blood pressure in PHPT. Both fasting and stimulated insulin values decreased following removal of the parathyroid tumor, but with no individual correlation with PTH and calcium levels.     

Biochemical characterization and localization of transglutaminase in wild-type and cell-death mutants of the nematode Caenorhabditis elegans

Glucose infusion into rats has been shown to sensitize/desensitize insulin secretion in response to glucose. In pancreatic islets from glucose-infused rats (GIR) (48 h, 50%, 2 ml/h) basal insulin release (2.8 mmol/l glucose) was more than fourfold compared with islets from saline-infused controls and the concentration-response curve for glucose was shifted to the left with a maximum at 11.1 mmol/l. The concentration-response curve for 45Ca2+ uptake was also shifted to the left in islets from GIR with a maximum at 11.1 mmol/l glucose. Starting from a high basal level at 2.8 mmol/l glucose KCl produced no insulin release or 45Ca2+ uptake in islets from GIR. Islets from GIR exhibited a higher ATP/ADP ratio in the presence of 2.8 mmol/l glucose and marked inhibition of 86Rb+ efflux occurred even at 3 mmol/l glucose. Moreover, in islets from GIR the redox ratios of pyridine nucleotides were increased. On the other hand insulin content was reduced to about 20%. The data suggest that a 48-h glucose infusion sensitizes glucose-induced insulin release in vitro in concentrations below 11.1 mmol/l. This may, at least in part, be due to enhanced glucose metabolism providing increased availability of critical metabolic factors including ATP which, in turn, decrease the threshold for depolarization and therefore calcium uptake. Calcium uptake may then be further augmented by elevation of the redox state of pyridine nucleotides.     

Does suppression of postprandial blood glucose excursions by the alpha-glucosidase inhibitor miglitol improve insulin sensitivity in diet-treated type II diabetic patients?

OBJECTIVE: Insulin sensitivity is impaired in patients with type II diabetes and is exacerbated by high mean blood glucose (BG). Potentially, large postprandial swings in BG could result in further decrements of insulin sensitivity. Because alpha-glucosidase inhibitors cause a marked reduction in the amplitude of BG changes, the aim of this study was to determine if such a BG-smoothing effect improves insulin sensitivity in well-controlled type II diabetic subjects treated with diet alone. RESEARCH DESIGN AND METHODS: Patients received either miglitol (BAY m 1099) (50 mg three times daily) or placebo for 8 weeks in a randomized double-blind parallel study. The miglitol (9 men, 2 women) and placebo (7 men, 3 women) groups were well matched (mean +/- SD) for age, weight, and blood glucose control (fasting BG, 6.4 +/- 1.0 vs. 6.9 +/- 1.6 mmol/l; HbA1, 7.7 +/- 1.0 vs. 7.9 +/- 0.4%; fructosamine, 0.99 +/- 0.08 vs. 1.07 +/- 0.17 mmol/l). The glucose metabolic clearance rate was calculated during the last 30 min of a 150 min glucose/insulin sensitivity test (glucose, 6 mg . kg-1 . min-1; insulin, 0.5 U . kg-1 . min-1). RESULTS: There was no significant improvement in metabolic clearance rate (0.21 +/- 0.27 vs. 0.16 +/- 0.35 l . kg-1 . min-1) for the miglitol- and placebo-treated groups, respectively. There were no statistically significant differences between miglitol and placebo for changes from baseline in BG (0.1 +/- 0.1 vs. -0.1 +/- 0.2 mmol/l), HbA1 (0.1 +/- 0.1 vs. 0.3 +/- 0.1%), and fructosamine (-0.06 +/- 0.02 vs. -0.03 +/- 0.02 mmol/l). CONCLUSIONS: Alpha-glucosidase-induced improvement in postprandial hyperglycemia does not result in increased insulin sensitivity.     

Altered insulin secretory responses to glucose in diabetic and nondiabetic subjects with mutations in the diabetes susceptibility gene MODY3 on chromosome 12

One form of maturity-onset diabetes of the young (MODY) results from mutations in a gene, designated MODY3, located on chromosome 12 in band q24. The present study was undertaken to define the interactions between glucose and insulin secretion rate (ISR) in subjects with mutations in MODY3. Of the 13 MODY3 subjects, six subjects with normal fasting glucose and glycosylated hemoglobin and seven overtly diabetic subjects were studied as were six nondiabetic control subjects. Each subject received graded intravenous glucose infusions on two occasions separated by a 42-h continuous intravenous glucose infusion designed to prime the beta-cell to secrete more insulin in response to glucose. ISRs were derived by deconvolution of peripheral C-peptide levels. Basal glucose levels were higher and insulin levels were lower in MODY3 subjects with diabetes compared with nondiabetic subjects or with normal healthy control subjects. In response to the graded glucose infusion, ISRs were significantly lower in the diabetic subjects over a broad range of glucose concentrations. ISRs in the nondiabetic MODY3 subjects were not significantly different from those of the control subjects at plasma glucose levels <8 mmol/l. As glucose rose above this level, however, the increase in insulin secretion in these subjects was significantly reduced. Administration of glucose by intravenous infusion for 42 h resulted in a significant increase in the amount of insulin secreted over the 5-9 mmol/l glucose concentration range in the control subjects and nondiabetic MODY3 subjects (by 38 and 35%, respectively), but no significant change was observed in the diabetic MODY3 subjects. In conclusion, in nondiabetic MODY3 subjects insulin secretion demonstrates a diminished ability to respond when blood glucose exceeds 8 mmol/l. The priming effect of glucose on insulin secretion is preserved. Thus, beta-cell dysfunction is present before the onset of overt hyperglycemia in this form of MODY. The defect in insulin secretion in the nondiabetic MODY3 subjects differs from that reported previously in nondiabetic MODY1 or mildly diabetic MODY2 subjects.     

Assessment of insulin sensitivity and secretion with the labelled intravenous glucose tolerance test: improved modelling analysis

A new modelling analysis was developed to assess insulin sensitivity with a tracer-modified intravenous glucose tolerance test (IVGTT). IVGTTs were performed in 5 normal (NGT) and 7 non-insulin-dependent diabetic (NIDDM) subjects. A 300 mg/kg glucose bolus containing [6,6-(2)H2]glucose was given at time 0. After 20 min, insulin was infused for 5 min (NGT, 0.03; NIDDM, 0.05 U/kg). Concentrations of tracer, glucose, insulin and C-peptide were measured for 240 min. A circulatory model for glucose kinetics was used. Glucose clearance was assumed to depend linearly on plasma insulin concentration delayed. Model parameters were: basal glucose clearance (Cl(b)), glucose clearance at 600 pmol/l insulin concentration (Cl600), basal glucose production (Pb), basal insulin sensitivity index (BSI = Cl(b)/basal insulin concentration); incremental insulin sensitivity index (ISI = slope of the relationship between insulin concentration and glucose clearance). Insulin secretion was calculated by deconvolution of C-peptide data. Indices of basal pancreatic sensitivity (PSIb) and first (PSI1) and second-phase (PSI2) sensitivity were calculated by normalizing insulin secretion to the prevailing glucose levels. Diabetic subjects were found to be insulin resistant (BSI: 2.3 +/- 0.6 vs 0.76 +/- 0.18 ml x min(-1) x m(-2) x pmol/l(-1), p < 0.02; ISI: 0.40 +/- 0.06 vs 0.13 +/- 0.05 ml x min(-1) x m(-2) x pmol/l(-1), p < 0.02; Cl600: 333 +/- 47 vs 137 +/- 26 ml x min(-1) x m(-2), p < 0.01; NGT vs NIDDM). Pb was not elevated in NIDDM (588 +/- 169 vs 606 +/- 123 micromol x min(-1) x m(-2), NGT vs NIDDM). Hepatic insulin resistance was however present as basal glucose and insulin were higher. PSI1 was impaired in NIDDM (67 +/- 15 vs 12 +/- 7 pmol x min x m(-2) x mmol/l(-1), p < 0.02; NGT vs NIDDM). In NGT and in a subset of NIDDM subjects (n = 4), PSIb was inversely correlated with BSI (r = 0.95, p < 0.0001, log transformation). This suggests the existence of a compensatory mechanism that increases pancreatic sensitivity in the presence of insulin resistance, which is normal in some NIDDM subjects and impaired in others. In conclusion, using a simple test the present analysis provides a rich set of parameters characterizing glucose metabolism and insulin secretion, agrees with the literature, and provides some new information on the relationship between insulin sensitivity and secretion.     

Comparative anatomy of arterial vascularization of the rhinencephalon in man, cat and sheep

Although the morphological development of the fetal pancreatic B cell has been studied in considerable detail, knowledge about the functional maturation, particularly in early stages of development, is still poor. The present paper describes a method for monolayer culture of fetal rat islet cells which allows a study of the regulation of insulin biosynthesis, release and content during critical stages of embryonic and fetal development. Suspensions of pancreatic cells were prepared from rat fetuses on pregnancy day 16 and cultured for 3 days. During the initial 2 days cultures were performed in the presence of 5 or 15 mmol/l glucose. During this initial period, culture at 5 mmol/l glucose was carried out in the presence or absence of either 10 mmol/l nicotinamide (NA) or 5 or 100 ng/ml nerve growth factor (NGF). After changing the media the cells were further exposed for 24 h to either 5 or 15 mmol/l glucose or 15 mmol/l glucose plus 5 mmol/l theophylline before measuring the insulin concentration in the culture medium. Cells that had initially been cultured for 2 days in 5 mmol/l glucose showed an increased insulin release, when subsequently cultured in 15 mmol/l glucose for 24 h. Theophylline potentiated the response and caused a decrease in cellular insulin content. Cells initially cultured in the presence of 15 mmol/l glucose showed unchanged insulin release during the subsequent 24-hour exposure to 15 mmol/l glucose, irrespective of the presence or absence of theophylline. The presence of NGF (100 ng/ml) during the initial 2-day culture period increased the insulin release in the presence of 15 mmol/l glucose and theophylline during the subsequent 24-hour culture period as compared to cells cultured in the absence of NGF. When cells were first exposed to either NA or NGF followed by exposure to 5 mmol/l glucose alone in the last 24-hour culture period, there was an increased insulin content. Rates of insulin biosynthesis remained unchanged irrespective of the glucose concentration in the culture medium. It is concluded that, already in early fetal development, B cells show glucose stimulation of insulin release albeit less pronounced than in the postnatal state.